Creatine kinase (E.C. 2.7.3.2 registered in International Union of Biochemistry) is an enzyme present in muscular tissues throughout the body and in the brain. In the field of clinical examinations, an assay of creatine kinase activity is one of the important examinations usually carried out for diagnosis of cardiac diseases, e.g., myocardial infarction, muscular diseases, e.g., progressive muscular aystrophy, nervous diseases, central nervous system diseases, mental disorders, and the like.
Creatine kinase is an enzyme which catalyzes the reversible reaction shown by the following scheme (1) in both directions: ##STR1## wherein ADP is adenosine diphosphate; and ATP is adenosine triphosphate.
Various methods have conventionally been proposed for assaying creatine kinase. One type of method comprises assaying the catalytic activity in the direction to the left of the above-described reaction (1). This type of method includes (a) a method of measuring an inorganic phosphoric acid released by hydrolysis of creatine phosphate, (b) a method comprising converting ADP to the oxidation of reduced from .beta.-nicotinamideadenine dinucleotide (hereinafter abbreviated as NADII) by the action of pyruvate kinase and lactate dehydrogenase and measuring the decrease in absorption at 340 nm due to the oxidation of NADH, (c) a method comprising converting ADP to pyruvic acid by the action of pyruvate kinase and measuring hydrazone produced by the reaction between pyruvic acid and 2,4-dinitrophenylhydrazine, and the like. In recent years, however, scarcely has any of those methods been employed, due to their low sensitivity or unstable color development. On the other hand, a method for assaying the activity of creatine kinase in the direction to the right of the above-described reaction (1) includes (d) a colorimetric or fluorometric method in which creatine produced is reacted with a dye, (c) a method of using luciferase as disclosed in Japanese Patent Application (OPI) Nos. 41597/76, 26200/81 and 105199/82 (the term "OPI" herein used means "unexamined published application") and Japanese Patent Publication No. 5678/83, (f) a method of using phosphoglycrerate kinase and glyceraldehyde-3-phosphate dehydrogenase as disclosed in Japanese Patent Publication No. 34119/84 and Japanese Patent Application (OPI) No. 155000/81, (g) a method of using hexokinase and glucose-6-phosphate dehydrogenase, and the like. Of these, the colorimetric or fluorometric method (d) has poor reliability on the measured values; the luciferase method (e) requires expensive luciferase and a specific apparatus for measurement; and the phosphoglycerate kinase/glyceraldehyde-3-phosphate dehydrogenase method (f) is an absorption decreasing system similar to the pyruvate kinase/lactate dehydrogenase method as described above, and, therefore, involves the same disadvantages as associated with the pyruvate kinase/lactate dehydrogenase method and, in addition, requires use of phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase that are more expensive than pyruvate kinase and lactate dehydrogenase. Hence any of the methods (d) to (f) is not satisfactory for practical use. The hexokinase/glucose-6-phosphate dehydrogenase method (g) has been employed most commonly because it is based on the most reasonable principle, exhibits satisfactory sensitivity and reproducibility and is capable of assaying a number of specimens. The principle of this assay method consists in the absorption increase at 340 nm due to the formation of reduced form .beta.-nicotinamideadenine dinucleotide (phosphate) which is finally produced by the following reaction schemes: ##STR2## wherein NAD(P) is .beta.-nicotinamideadenic dinucleotide (phosphate); and NAD(P)H is reduced form .beta.-nicotinamideadenine dinucleotide (phosphate).
Ever since the first report on the hexokinase/glucose-6-phosphate dehydrogenase method by T.T. Oliver in Biochem. J., Vol. 61, pp. 116-122 (1955), various improvements have been made. For example, there have been conducted studies on an assay method for inhibiting an activity of adenylic kinase that mainly exists in blood and causes a negative error in the hexokinase/glucose-6-phosphate dehydrogenase method, as described in U.S. Pat. No. 4,220,714, European Pat. No. 71087 (corresponding to Canadian Pat. No. 1,175,737), G. Szasz, W. Gerhardt, W. Gruber and E. Bernt, Clin. Chem., Vol. 22, pp. 1806-1811 (1976) and G. Szasz, W. Gerhardt and W. Gruber, Clin. Chem., Vol. 23, pp. 1888-1892 (1977); studies on thiol compounds for activation of creatine kinase as described in Japanese Patent Application (OPI) No. 106397/74 (DE 2302721) and G. Szasz, W. Gerhardt and W. Gruber, Clin. Chem., Vol. 24, pp. 1557-1563 (1978); studies on the use of chelate compounds and stability of a reagent for assaying creatine kinase activity as described in G. Szasz, J. Waldenstrom, and W. Gruber, Clin. Chem., Vol. 25, pp. 446-452 (1979); and the like. As a result, the hexokinase/glucose-6-phosphate dehydrogenase method has been established as the most reliable assay method for creatine kinase in clinical laboratories.
Nevertheless, the hexokinase/glucose-6-phosphate dehydrogenase method still has problems awaiting solution with respect to analytical accuracy and stability of a reagent for assaying creatine kinase. The former problem comes from the possible action of hexokinase on sugars other than glucose existing in body fluids, such as fructose and mannose, which results in a positive error of measured values. The latter problem is ascribed to the poor stability of the reagent during preservation in the form of a solution, i.e., a short working life of the reagent in a liquid state at room temperature (18.degree. to 35.degree. C.), even if a stabilizer, such as phycol (as described in Japanese Patent Application (OPI) No. 12897/77) and albumin, is added. Moreover, even in a so-called two-reagent system wherein the reagent is divided into two containers in a pH region of from 6.5 to 7.0, the stability of the reagent cannot be improved as desired so as to withstand use for a prolonged period of time in clinical laboratories. Therefore, satisfactory solutions to these problems have been strongly desired.
In order to overcome the above-described disadvantages encountered in the hexokinase/glucose-6-phosphate dehydrogenase method, a one-reagent system using glucokinase having extreme specificity to glucose and excellent heat stability has been described to be used in place of hexokinase, as disclosed in U.S. Pat. No. 4,438,199 (corresponding to European Patent Publication No. 43181A and Japanese patent application (OPI) No. 169598/81) and U.S. patent application Ser. No. 580,503 (corresponding to European Patent Publication No. 119722A and Japanese Patent Application (OPI) No. 151899/84). This glucokinase/glucose-6-phosphate dehydrogenase method also improves stability of a reagent for assaying creatine kinase in terms of preservation in a dissolved state at room temperature.
Although the glucokinase/glucose-6-phosphate dehydrogenase method somewhat resolved the problem of instability of the reagent after dissolution that was associated with the aforesaid hexokinase/glucose-6-phosphate dehydrogenase method, the stability of the reagent was still insufficient and a relatively large quantity of enzymes was required for maintaining the stability of the reagent for extended periods of time. In addition, with the recent increase of diseases that need urgent assays in clinical laboratories, such as myocardial infaraction there has been a pressing demand for development of a reagent which enables accurate and rapid determination of creatine kinase activity in vital body fluids. In other words, high stability in the form of a solution for a long period of time has been required of a reagent so that creatine kinase activity can be assayed in any time of emergency without requiring adjustment of a reagent for minimizing measurement errors.